The permissive environment of the fetus allows a unique opportunity to engraft allogeneic or xenogeneic hematopoietic stem cells (HSC). Transplantation of HSC in utero has tremendous potential clinical application toward the treatment of prenatally diagnosed fetuses with hematopoietic disease, including hemoglobinopathies, immunodeficiency syndromes, and inborn errors of metabolism. Our long-term objective is the optimal clinical application of fetal HSC transplantation for prenatal treatment of fetuses diagnosed with hematopoietic disease. We have previously confirmed the promise of fetal HSC transplantation in large animal allogeneic and xenogeneic models by the successful creation of long-term, multilineage, hematopoietic chimerism. However, large animal models are inherently limited by long gestation, cost and immunologic complexity. In contrast, the successful development of a rodent model would allow a large number of experiments to be performed in a relatively short period of time at minimal cost. In addition, the availability of inbred strains and murine models of human disease will allow investigation of the immunobiology of prenatal HSC transplantation, as well as experimental analysis of the prenatal treatment of analogous murine diseases. In this application, we propose the development and characterization of a murine model of fetal HSC transplantation to facilitate efficient, and cost effective optimization of the prenatal approach. Specifically, we propose the creation of hematopoietic chimerism by the in utero transplantation of HSC in the mouse model using recently established techniques of magnification, transillumination, and micropipette injection. We will use specific and sensitive detection methodology to confirm and characterize engraftment and will evaluate requirements for optimal engraftment. Because the primary limitation to clinical application of the prenatal approach is limited donor cell engraftment, we will focus this proposal on various strategies to improve donor cell engraftment and expression. Strategies will include increasing donor HSC dose by multiple injections or use of donor cell preparations enriched for HSC, or cytokine manipulations of donor cells and/or the recipient. Successful development of the murine model will greatly accelerate our progress in prenatal HSC transplantation and direct confirmatory studies in large animal models prior to clinical application.